SK7342002A3 - Process for recovering statin compounds from a fermentation broth - Google Patents

Abstract

A novel process for recovering a compound from a fermentation broth that includes the stages of forming an enriched solution of the compound by extraction, obtaining a salt of the compound from the enriched solution, purifying a salt of the compound and trans-salifying the salt of the compound to a metal salt of the compound is disclosed.

Description

Method for isolating statin compounds from fermentation broth

Technical field

The invention relates to methods of isolating desired chemical products of reactions carried out in aqueous fermentation broth. The invention further relates to the isolation of pravastatin, compactin and lovastatin from the fermentation broth, and in particular to the isolation of pravastatin prepared by fermentation of compactin.

BACKGROUND OF THE INVENTION

Complications of cardiovascular disease such as myocardial infarction, stroke and peripheral vascular disease are attributable to half the deaths in the United States. The high level of low-density lipoprotein (LDL) in the bloodstream is associated with coronary lesions that prevent blood flow and can rupture and cause thrombosis. Goodman and Gilman, The Pharmacological Basis of Therapeutics 879 (9th edition, 1996). Reduction of plasma LDL levels in patients with cardiovascular disease and patients who, although not suffering from cardiovascular disease but suffering from hypercholesterolaemia, has been shown to reduce the risk of clinical events. Scandinavian Simvastatin Survival Study Group (1994); Lipid Research Clinics Program, 1984a, 1984b.

Statin drugs are currently the most therapeutically effective drugs available to reduce LDL levels in the bloodstream of a patient at risk of cardiovascular disease. This class of drugs includes, but is not limited to, compactin, lovastatin, simvastatin, pravastatin and fluvastatin. The mechanism of action of statin drugs has been clarified to some extent. Statins interfere with the synthesis of cholesterol $ other sterols in the liver by competitively inhibiting 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMG-CoA reductase). HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevalonate, a step that determines the rate of cholesterol biosynthesis. Its inhibition therefore leads to a decrease in the rate of cholesterol formation in the liver.

Pravastatin is the generic name of the chemical compound [1S- [Ια (β *, δ *) 2α, βα, 8β (R *), 8aa]] -1, 2,6,7,8,8a-hexahydro-p, δ, 6-trihydroxy-2-methyl-8- (2-methyl-1-oxobutoxy) -1-naphthalene-heptanoic acid (CAS Registry 81093-370). The molecular structure of pravastatin in the free acid form corresponds to the general formula Ia, wherein R is hydroxy. The lactone form is represented by the general formula Ib, in which the numbering of the atoms is indicated.

(Ib)

Pravastatin, compactin (a compound of formula Ib wherein R is hydrogen), lovastatin (a compound of formula Ib wherein R is CH ₃ ), simvastatin and fluvastatin each contain an alkyl chain terminated in a lactone-terminated carboxylic acid group and carrying two hydroxy groups in the β and a positions relative to the carboxylic acid group. This alkyl chain is that part of the molecule that binds to HMG-CoA reductase. The carboxylic acid group and the hydroxy group at position b tend to be lactonized (see Formula Ib). Lactonizable compounds, such as statins, may be in the free acid or lactone form, or as an equilibrium mixture of both. Lactonization is a source of processing difficulties in the manufacture of statin drugs because the free acid form and the lactone form have different polarities. Cleaning of one mold is likely to remove the other mold together with impurities, resulting in lower yields. It is therefore usually necessary to proceed very carefully when processing the lactonizable compounds to isolate them in high yield.

Pravastatin has an important therapeutic advantage over other statins. Pravastatin selectively inhibits the synthesis of cholesterol in the liver and small intestine, but does not substantially affect cholesterol synthesis in peripheral cells. Koga, T. et al., Biochem. Biophys. Acta, 1990, 1045, 115-120. This selectivity is probably due in part to the presence of the hydroxy group at the 6-position of the hexahydronaphthalene core. The C-6 position is occupied by a hydrogen atom in the compactin and by a methyl group in lovastatin. Pravastatin has a lower ability to cross the lipophilic membranes of peripheral cells than other lipophilic congeners, Serajuddin et al., J. Pharm. Sci., 1991, 80, 830-834, and a more localized action in the liver and intestine is attributed to the limited mobility of pravastatin.

According to U.S. Pat. No. 4,346,227, which is incorporated herein by reference, pravastatin was first isolated as a compactin metabolite by M. Tanaka et al. during the study of compactin metabolism. According to this patent, pravastatin can be obtained by fermentation of compactin using various microorganisms: Absidia coerulea IFO 4423 spores, Cunninghamella echinulata IFO 4445, Streptomyces rosochromogenus NRRL 1233, Syncephalas trum racemosum IFO 4814 and Syncephalastrum racemosum IFO 4828 fermented after fermentation after fermentation by fermentation. If the soil is acidified to pH 3, pravastatin and other non-hydrophilic organics are extracted with ethyl acetate and the extract is washed with aqueous sodium chloride solution. The free acid pravastatin is lactonized by adding a catalytic amount of trifluoroacetic acid, neutralizing with dilute sodium bicarbonate, drying over sodium sulfate and evaporating to dryness. The residue was purified by reverse phase preparative high performance liquid chromatography (HPLC). It will be apparent to one skilled in the art that reverse phase HPLC in the large-scale production of chemical compounds is not an economical purification method.

U.S. Pat. No. 5,942,423 relates to microbial hydroxylation of compactin to pravastatin using the Äctinomadura strain. The only isolation method given in the examples is the isolation of minute amounts resulting from the analytical scale in which the HPLC analysis of the fermentation broth is carried out. As is clear from the more general discussion of the isolation of pravastatin from the fermentation broth, the preferred method of isolation is by HPLC.

PCT application no. PCT / U500 / 19384 relates to microbial hydroxylation of compactin to pravastatin using a strain of Micromonospora maculata that is unusually resistant to the antifungal effects of compactin.

U.S. Pat. No. 5,202,029 relates to a method for purifying HMG-CoA reductase inhibitors using HPLC. After separation of impurities on the HPLC column, the HMC-CoA reductase inhibitor is eluted from the HPLC column as a substance dissolved in the eluent. The eluent is partially evaporated and the addition of water induces crystallization of the HMC-CoA reductase inhibitor.

U.S. Pat. No. 5,616,595 relates to a continuous process for the isolation of water-insoluble compounds from a fermentation broth by tangential filtration. The fermentation broth is circulated through a filter. With each cycle, the concentration of the fermentation broth increases because of the loss of water during filtration. After reaching the desired concentration, the concentrated fermentation broth is suspended using a solvent in which the desired compound is soluble. The resulting suspension was circulated through a filter. A solution of the desired compound is collected as a filtrate, which is isolated from the filtrate and optionally subjected to further purification. This method is said to be applicable to a variety of compounds including lovastatin, pravastatin and simvastatin.

A method for isolating lovastatin in the form of lactone is described in U.S. Pat. In this method, lovastatin is extracted from the fermentation broth with butyl acetate. The resulting solution is then centrifuged and the separated aqueous phase is discarded. The organic phase is subjected to vacuum distillation at a temperature above 40 ° C, which (in addition to concentrating the solution) promotes lactonization by removal of water. Upon cooling, lovastatin lactone crystallizes, which after recrystallization has a purity of 90% or more. One skilled in the art will appreciate that this method is not suitable for isolating a statin in the form of a free carboxylic acid or in the form of a carboxylate salt.

At present, it is economically most advantageous to prepare pravastatin by enzymatic hydroxylation of the C-6 compactin. However, the known methods of isolating a statin from a fermentation broth are not suitable for isolating pravastatin in the form of the sodium salt, do not achieve a pharmaceutically acceptable level of purity, or chromatographic separation is necessary to achieve high purity.

The solution according to the invention meets the need in the art for an efficient process for the isolation of pravastatin from a fermentation broth on a preparative scale, which yields the product in high yield and high purity without the need for chromatographic purification.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION It is an object of the present invention to provide an efficient method for isolating a statin compound from an aqueous fermentation broth, in particular a process for purifying pravastatin, compactin and lovastatin on an industrial scale without the need for chromatographic separation.

Another object of the invention is to obtain pravastatin in a highly pure form and in a high yield so as to achieve significant stereoselectivity and regioselectivity of microbial transformations, as well as higher yields and greater economy. Pravastatin is isolated from the soil with minimal solvent consumption, purified in high yield, and converted to its pharmaceutically acceptable sodium salt.

In this method, pravastatin is extracted from the aqueous fermentation broth into an organic solvent, then back-extracted into a basic aqueous solution and optionally subsequently re-extracted into an organic solvent or the aqueous solution is concentrated to obtain an aqueous or organic solution which is relative to the initial pravastatin concentration in fermentation broth enriched with pravastatin. Pravastatin is obtained from the enriched solution by precipitation in the form of a metal salt or an ammonium salt, and this salt is then purified by recrystallization. The recrystallized salt is transsalified to give pravastatin sodium and any excess sodium ions are captured with an ion exchange resin. The pravastatin sodium can then be isolated from the solution by recrystallization, lyophilization, or otherwise.

A more detailed description of the invention follows.

The present invention provides a process for the isolation of pravastatin, compactin and lovastatin from an aqueous fermentation broth. The invention is illustrated to isolate pravastatin sodium from a fermentation broth. However, this method can also be used to purify other compounds prepared by microbial or enzymatic methods.

Enzymatic hydroxylation of compactin

Pravastatin sodium is synthesized by enzymatic hydroxylation of compactin as described in U.S. Pat. The hydroxylation soil from which pravastatin is to be isolated can be any aqueous soil known for compactin fermentation on an industrial scale. If the soil is neutral or basic after the fermentation is complete, acid is added thereto to adjust the pH of the soil to about 1 to about 6, preferably about 1 to about 5.5, and more preferably to 2 to 4. As the acid that is mention may be made of hydrochloric acid, sulfuric acid, trifluoroacetic acid or any other protic acid, preferably an acid whose IM solution in water has a pH of less than 1. By acidifying the fermentation broth, all pravastatin carboxylate salts are converted to the free acid and / or or lactone.

Isolation of pravastatin sodium

The process of the invention comprises the step of forming an enriched pravastatin solution, the step of recovering the pravastatin salt from the enriched solution, the step of purifying the pravastatin salt, the step of transsalifying the pravastatin salt into pravastatin sodium and the step of isolating pravastatin sodium.

In the first step, pravastatin is recovered from the aqueous fermentation broth in a relatively highly concentrated solution by a sequence of extraction and back extraction operations. Fermentation is typically carried out at very high dilution. By dilution the soil achieves a higher maximum enzymatic potential. The disadvantage of high dilution is that a large volume of fermentation medium must be handled until the desired product is obtained in a more enriched form. The large volume also imposes strict requirements on the method of insulation. To separate such large volumes, chromatographic procedures are usually impractical, especially when the solvent is water. If the isolation is carried out by extraction, the organic solvent must have sufficient polarity to compete with water for a favorable distribution of the product, but must not be sufficiently polar to be substantially water soluble. If the extraction is inefficient, isolation of the desired product in high yield requires the use of large volumes of organic solvent, which entails health and safety risks for personnel working with or near the fermenter.

We have found that C 2 -C 4 alkyl formates and C 1 -C 4 alkyl esters of C 2 -C 4 carboxylic acids are capable of highly extracting pravastatin from an aqueous fermentation broth. The partition coefficient of pravastatin lactone is typically 1000: 1 or higher and the partition coefficient of free acid is 10: 1 or higher. The alkyl groups may be linear, branched or cyclic. Preferred esters include ethyl formate, n-propyl formate, i-propyl formate, n-butyl formate, s-butyl formate, i-butyl formate, t-butyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, s-butyl acetate, isobutyl acetate, tert-butyl acetate, methylpropionate, ethylpropionate, n-propylpropionate, i-propylpropionate, butylpropionate, methylbutyrate, ethylbutyrate, n-propylbutyrate, i-propylbutyrate, butylbutyrates, methylisobutyrate, ethylisobutyrate and propylisobutyrate, propylisobutyrate. Among these preferred organic solvents, we have found that ethyl acetate, i-butyl acetate, propyl acetate and ethyl formate are particularly suitable. The most preferred extraction solvent is i-butyl acetate. Other organic solvents such as halogenated hydrocarbons, aromatic compounds, ketones and ethers can be used instead of esters. Examples thereof include dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, benzene, butylmethylketone, diethyl ether and methyl tert-butyl ether.

In the enrichment step of the process according to our invention, an organic extract is formed by contacting the organic extraction solvent, preferably selected from the above list, with the fermentation broth. The pH of the fermentation broth is from about 1 to about 6, preferably from about 2 to about 4.

Any apparatus adapted to mix large volumes of liquids in a batch or continuous process may be used. Since the fermentation is typically carried out as a batch, the batch method of isolation is a natural choice. Thus, it is possible to use conventional large-volume mixers and settling tanks or equipment adapted for both mixing and phase separation. In a preferred embodiment of this aspect of the invention, a minor part, preferably less than 50% by volume, of the extraction solvent is contacted with the fermentation broth, preferably with gentle mechanical stirring. After contacting and phase separation, pravastatin-containing extraction solvent is separated from pravastatin depleted fermentation broth. The soil may be contacted with the organic extraction solvent one or more times, and any resulting organic extracts may be combined. The volume of the resulting pravastatin-containing organic extract may be greater or less than the volume of the fermentation broth.

A second operation leading to the formation of an enriched pravastatin solution is the back extraction of pravastatin into a basic aqueous solution. Back-extraction removes some or all of the non-polar organic impurities and, if pravastatin-lactone is present, back-extraction promotes re-opening of its lactone ring. Without wishing to be limited in any way by a particular chemical theory or mechanism, according to established chemical theory, pravastatin is in the basic aqueous extract in the form of a carboxylate anion. Back extraction can be used to concentrate pravastatin by using an aqueous base in a volume less than the volume of the organic extract. The base is preferably sodium hydroxide, ammonium hydroxide or potassium hydroxide, preferably ammonium hydroxide or sodium hydroxide, and the basic aqueous solution preferably has a pH in the range of about 7.0 to about 13.7, more preferably from about 7 to about 13, and most preferably from about 7 to about 13.7. about 7.5 to about 11. The extraction solvent is contacted with the basic aqueous solution until the amount of pravastatin in the organic phase is substantially depleted, as determined by thin layer chromatography or any other procedure, including subjective assessment of the occurrence. contact sufficient for complete extraction. Multiple back extraction can be performed for optimal isolation. However, when the organic phase is butyl acetate, a single back extraction is highly efficient. Preferably, the back extraction is carried out using a basic aqueous solution in a volume that is less than one-third the volume of the organic extract, preferably less than one-quarter, and most preferably is about one-fifth the volume of the organic extract. The concentration of the enriched aqueous solution from which pravastatin is later obtained is preferably in the range from about 2 to about 50 g / liter, more preferably from about 5 to about 15 g / liter.

The aqueous extract can further be concentrated by distillation, preferably by vacuum distillation, to increase the concentration of the solution. Before further concentrating the aqueous extract by distillation, the pH should be adjusted to about 7 to about 13.7, preferably to about 7.5, to about 11, and more preferably to about 8 to about 10. Vacuum distillation may be carried out such that the aqueous extract is is heated to a temperature of from about 30 ° C to about 80 ° C under an absolute pressure of 10 to 10 mbar. The choice of other vacuum distillation conditions is within the ability of those skilled in the art to which this process pertains.

An alternative to obtaining pravastatin from an enriched aqueous solution later in the process of the invention is the possibility of obtaining pravastatin from an enriched organic solution. The enriched organic pravastatin solution is obtained by reextracting pravastatin into an organic solvent after the aqueous extract has been acidified again with an acid, preferably trifluoroacetic acid, hydrochloric acid, sulfuric acid, acetic acid or phosphoric acid, preferably sulfuric or phosphoric acid, to a pH of about 1.0 to about 6.5, more preferably from about 2.0 to about 4.0. Depending on the conditions, the carboxylate anion of pravastatin can be protonated to pravastatin - a free acid that is less polar than the carboxylate, or lactonized to an even less polar form.

Pravastatin is re-extracted into a re-extraction solvent selected from the organic solvents listed above as suitable for extraction of pravastatin from the fermentation broth. The organic solvent may or may not be the same as the solvent used to extract pravastatin from the fermentation broth. In this reextraction, further enrichment of pravastatin by reextraction to an amount of organic solvent is achieved, which is preferably less than about 50% by volume of the aqueous extract, more preferably about 33% to about 20%, and even more preferably about 25% by volume of the aqueous extract. Thus, as further illustrated in Example 1, pravastatin can be concentrated from 100 liters of fermentation broth to 8 liters of enriched organic solution in 89% yield based on the initial organic extract. However, one skilled in the art will be able to achieve a higher yield of purified pravastatin by multiple extraction. However, only the preferred embodiment of single extraction has been described above. In this preferred embodiment, a balance is achieved between solvent costs and high product yield. Deviations from this preferred process, which achieve further improvement in yield by repeated extractions and from which only one embodiment is described above, do not necessarily represent a departure from the spirit of the invention. Prior to treatment to obtain pravastatin from the enriched organic solution by salting out, the enriched organic solution is preferably dried, which may be accomplished using conventional drying agents such as magnesium sulfate, sodium sulfate, calcium sulfate, silica, perlite and the like, and optionally decolourised with charcoal. The dried and / or decoloured enriched organic solution is then separated using conventional methods, for example by filtration or decantation.

In the next step of the process according to the invention, the pravastatin salt is obtained from the enriched aqueous or organic solution.

The salt is obtained by precipitation from the enriched solution.

Precipitation is induced by adding a metal salt, ammonia, amine, ammonium salt or amine salt to the enriched solution.

Metal salts which may be used include hydroxides, alkoxides, halides, carbonates, borates, phosphates, thiocyanates, acetates, nitrates, sulfates, thiosulfates and any other salts that are highly soluble in water. Metals include lithium, sodium, potassium, calcium, magnesium, copper, iron, nickel, manganese, tin, zinc, and aluminum. Preference is given to salts containing the following metal cations: Li ⁺ ,

Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Cu ²⁺ , Fe ²⁺ , Fe ³⁺ , Ni ²⁺ , Mn ²⁺ , Sn ²⁺ , Zn ²⁺ and Al ³⁺ .

In salts which induce the precipitation of pravastatin salt with a metal, sodium and potassium are most preferred as the metal cation.

Pravastatin can also be precipitated as an ammonium or amine salt by addition of ammonia or amine. The amine may be a primary, secondary or tertiary amine. Any alkyl or arylamine which is not hindered in such a way as to prevent ionic interactions between the amine nitrogen and the carboxy group of pravastatin can be used. Non-limiting examples of amines include methyl-, dimethyl-, trimethyl-, ethyl-, diethyl-, triethylamine and other primary, secondary and tertiary amines having 1 to 6 carbon atoms in each of the hydrocarbon residues, and morpholine, N-methylmorpholine. , isopropylcyclohexylamine, piperidine and the like. Regardless of the absence, presence or multiple substitution at the nitrogen, salts formed by reaction with ammonia or amine are hereinafter referred to as ammonium salts. Thus, the term both salts with amines and salts with ammonia are meant.

The ammonium salt of parvastatin can also be precipitated by the addition of an ammonium salt, either alone or in combination with ammonia, an amine or a metal salt. Preferred are the following ammonium salts: ammonium chloride, NH ₄ Cl; ammonium bromide,

NH ₄ Br; ammonium iodide, NH ₄ I;

ammonium sulfate, (NH ₄ ) ₂ SO ₄ ; ammonium nitrate, NH ₄ NO ₃ ; ammonium phosphate, (NH ₄ ) ₃ PO ₄ ; ammonium sulfite, (NH ₄ ) ₂ S ₂ O ₄ ;

ammonium acetate,

NH ₄ OAc and ammonium thiocyanate, NH ₄ SCN. Most preferred is ammonium chloride.

Metal salts, ammonium salts and high boiling liquid and solid amines can be added by conventional means, preferably in well ventilated areas, either as solids, liquids in substance or solutions in aqueous or organic solvents. The addition of ammonia gas requires special equipment for handling caustic gases. Equipment such as pressure vessels, regulators, valves and piping are readily available. Ammonia can be introduced into the upper space above the enriched solution at ambient pressure or, if pressure vessels are used, at elevated pressure. Alternatively, ammonia may be bubbled through a solution that is preferably stirred to reduce clogging of the feed tube with precipitated pravastatin ammonium salt.

In a preferred embodiment of the process according to the invention, pravastatin is obtained from the enriched solution as an ammonium salt by addition of ammonia or an amine. In a more preferred embodiment, pravastatin is obtained from the enriched solution in the form of an ammonium salt by adding ammonia gas to the enriched solution. Using ammonia, the highly polar ammonium salt of pravastatin is obtained, which precipitates easily and in high yield from the anti-solvent. In the most preferred embodiment, pravastatin is obtained as the ammonium salt of pravastatin by addition of gaseous ammonia and an ammonium salt. The most preferred ammonium salt is ammonium chloride, the advantage being that a concentrated aqueous ammonium chloride solution is formed in the event of incomplete drying of the enriched organic solution.

The temperature at which a metal salt, ammonia, amine and / or ammonium salt should be added can be determined by routine small scale reactions monitoring its exothermic properties. The preferred embodiment will not allow the temperature of the solution to rise above 40'C. Although temperatures as high as 80 ° C may be used without significantly decomposing pravastatin, many of the solvents used for the purposes of the present invention will boil at lower temperatures. When ammonia is used, the temperature is preferably in the range of about -10 to about 40 ° C.

As soon as the precipitation stops or it is determined by other means that pravastatin has been substantially consumed, the addition should be terminated. When ammonia or volatile amine is used, the container should be ventilated to dissipate excess vapors. The crystals are then isolated by filtration, decanting the solvent, evaporating the solvent or otherwise, preferably by filtration.

After optionally washing the precipitated crystals, the pravastatin salt is purified by single or multiple recrystallization. For purification, the pravastatin salt is first dissolved in water. Preferably a minimum amount of water is used. If an amine salt has been obtained instead of a metal or ammonia salt, more water will be required. Upon complete dissolution of the pravastatin salt, the polarity of the solution is reduced by the addition of an anti-solvent. The anti-solvent is a water-soluble organic solvent or solvent mixture in which pravastatin is poorly soluble. Suitable water-soluble organic solvents include acetone, acetonitrile, alkyl acetates, isobutanol and ethanol.

The pravastatin salts can be recrystallized spontaneously or induced by recrystallization by performing additional steps such as addition of a common ion, cooling or addition of seed crystals. The induced recrystallization by addition of a common ion is carried out by adding to the mixture a salt containing the same metal ion or ammonium ion as contained in the pravastatin salt. Suitable salts for inducing recrystallization of pravastatin salts are metal salts and ammonium salts which can be used to precipitate pravastatin salt from an enriched solution. In a preferred process where pravastatin is obtained as an ammonium salt, the chloride salt used with ammonia or amine is used first to induce recrystallization of the pravastatin salt. In the most preferred embodiment, in which the ammonium salt of pravastatin is obtained, the addition salt is most preferably ammonium chloride.

Recrystallization can be performed at about -10 ° C to about 60'C, preferably at about 0 to about 50'C, and most preferably at about 0 to about 40'C. After the pravastatin salt is substantially recrystallized from the solution, the crystals are isolated and can be washed, for example, with a 1: 1 mixture of isobutyl acetate and acetone and dried. The drying may be carried out at ambient temperature, but preferably at a slightly elevated temperature below about 45'C, preferably at about 40'C. In order to obtain a good effect, as in the processes of Examples 7 and 8, recrystallization is optionally repeated. Each repetition is carried out with a yield of 92 to 96%.

Even after recrystallization, pravastatin contains an organic impurity with a relative HPLC retention time of 0.9. Based on the HPLC chromatogram obtained using UV detection, this organic impurity is assumed to constitute about 0.2% of the total product. The organic impurity can be removed as follows.

The pravastatin salt is dissolved in water, preferably in a minimum amount or using a volume of 6 ml / g. About 0.2% by volume of isobutanol is added to the resulting solution. The pH of the mixture is increased to about 8 to about 14, preferably about 10 to about 13.7 by the addition of sodium hydroxide, and the mixture is maintained at about 10 to about 50 ° C for 10 to 200 minutes, preferably at about 20 to about 20 ° C. about 30 ° C for 60 to 100 minutes. The resulting solution is again acidified with a mineral or organic acid, preferably hydrochloric or sulfuric acid, to a pH of about 4 to about 9, more preferably about 5 to about 9, and most preferably about 6 to about 7.5. After pH adjustment, ammonium chloride is added to the solution to salt the pravastatin salt. If water is used in an amount of about 6 ml / g, it is recommended to add about 2.0 to 2.3 g of ammonium chloride per gram of pravastatin salt. The ammonium chloride is preferably added in portions over 4 to 6 hours.

Upon addition of ammonium chloride, the ammonium salt of pravastatin can crystallize spontaneously. Alternatively, recrystallization can be induced by cooling, seeding, or other conventional means. Although recrystallization is preferably induced by the addition of a common ion, for example by the addition of ammonium chloride, it can also be induced by dilution with an anti-solvent as in the recrystallization step described above. However, this operation should be carried out with care to avoid accidental precipitation of pravastatin salts. As described in more detail in Example 1, the amount of organic impurity with a relative retention time of 0.9 is reduced below the detectable limit and the pravastatin ammonium salt is obtained in about 99.3% purity to achieve a purity acceptable in pharmaceutical use. At this stage of the process of the invention, it is possible to obtain an ammonium salt of pravastatin which contains less than about 0.7% by weight of organic impurities.

After removal of the organic impurities by recrystallization, the pravastatin salt is transsalified to the pravastatin sodium. Transsalification refers to the process by which a cation of an organic salt molecule is exchanged for another. In transsalification, pravastatin is first released from its metal salt or ammonium salt by dissolving the salt in an aqueous solvent, adding any protic acid such as hydrochloric, sulfuric, phosphoric, trifluoroacetic or acetic acid to the aqueous solution, and pravastatin from the aqueous solution extracted with an organic solvent. The protic acid is added to the aqueous solution in an amount that neutralizes or acidifies it, preferably acidified to a pH of about 1 to about 6, more preferably about 2 to about 4. The aqueous solution is contacted with or before the protic acid is added to the aqueous solution. a water-immiscible organic solvent such as isobutyl acetate or any other water-immiscible organic solvent. After the aqueous solution is contacted with a water-immiscible organic solvent and mixed with a protic acid, the organic phase containing pravastatin is separated from the aqueous phase and after optionally washed with water to remove ammonium residues, pravastatin is back-extracted with aqueous sodium hydroxide. Preferably sodium hydroxide is used only in a slight molar excess relative to the amount of pravastatin, preferably less than 1.1 equivalents, more preferably less than 1.02 equivalents. After extraction into aqueous sodium hydroxide, the excess sodium cations are trapped to achieve a 1: 1 equivalence of sodium cations and pravastatin. The capture is performed using a water insoluble ion exchange resin. Suitable ion exchange resins are those of the cationic and chelate type, preferably strongly and weakly acidic ion exchangers.

Strongly acidic cation exchange resins that can be used include cation exchangers containing sulfonic acid groups (SO ₃ "H ⁺ ). Examples include the commercial products of Amberlite® IR-118, IR-120, 252H; Amberlyst (R) 15, 36; Amberjet® 1200 (H) (Rohm &Haas); Dowex® 50WX Series, Dowex® HCR-W2, Dowex® 650C, Dowex® Marathon C, Dowex® DR-2030 and Dowex® HCR-S, ion exchange resins (Dow Chemical Co.); Diaion® SK 102-116 series (Mitsubishi Chemical Corp.) and Lewatit SP 120 (Bayer). Preferred strongly acidic cation exchange resins are Amberlite® 120, Dowex® 50 WX and Diaion® SK series.

Low acid cation exchange resins include those containing pendant carboxylic acid groups. The weakly acidic cation exchange resins include the commercial products Amberlite® CG-50, IRP-64, IRC 50 and C 67, the Dowex® CCR series, the Lewatit® CNP series and the Diaion® WK series, the most preferred of which are Amberlite® resins. IRC50, Lewatit® CNPBO and Diaion® WK 10. Less preferred are ion exchange resins of the chelate type. Some of the types available on the market include Duolite® C-718 and C-467 (Rohm & Haas).

The solution containing pravastatin sodium and excess sodium cations can be contacted with the ion exchange resin by any method known in the art, for example by passing the solution through a column or bed of resin or by mixing the solution in the vessel with sufficient resin. The method of contact is not critical. After capture of excess sodium ions, the pH of the pravastatin sodium solution should be in the range of about 6.5 to about 10, preferably about 7.4 to about 7.8, although the pH will depend on dilution. Lowering the pH of the pravastatin sodium solution from higher to lower and stabilizing it at a lower value is evidence that the capture of excess sodium ions is substantially complete. After the capture is complete, the pravastatin sodium solution is separated from the resin in the usual manner. The solution may be collected as an eluate from a column or bed, or it may be filtered, decanted, and the like.

Pravastatin sodium can be isolated from the solution of pravastatin sodium by crystallization.

Effective crystallization may first require partial removal of water, which may be accomplished by vacuum distillation or nanofiltration. In a preferred embodiment, the aqueous solution of pravastatin sodium is concentrated to about 20 to about 50% (w / v) prior to crystallization. If desired, after concentration of the aqueous pravastatin sodium solution, the pH can be adjusted to about 7 to about 10 using an ion exchange resin in H ⁺ form.

Crystallization may be aided by the addition of a water-soluble organic solvent or organic solvent mixture. In this connection, mention may be made in particular of acetone and mixtures of acetone and acetonitrile, ethanol and acetonitrile, and ethanol and ethyl acetate. One of the most preferred solvent systems for crystallizing pravastatin sodium is a 1: 3: 12 mixture of water, acetone and acetonitrile, which is formed by concentrating the pravastatin sodium solution to about 30% (w / v) and then an appropriate volume of a 1: 4 mixture of acetone and acetonitrile is added thereto. Another very suitable crystallization solvent mixture is a 1: 15 mixture of water and acetone.

Pravastatin sodium can also be isolated by lyophilizing an aqueous solution of pravastatin sodium.

When the isolation is carried out by crystallization or other methods that improve the purity of the product, the pravastatin sodium isolated in the practice of this invention is substantially free of pravastatin lactone. As shown in the examples below, it is possible to isolate pravastatin sodium containing less than 0.5 wt. total impurities. Using the preferred embodiments of the invention, two of which are described in Examples 1 and 15, it is possible to isolate pravastatin sodium containing 0.2 wt. or less total impurity. The main impurities involved in the total impurity are epipravastine sodium, 3'-OH compactin sodium, 6-hydroxyisocompactin sodium and pravastatin lactone.

The invention is illustrated by the following examples. These examples are illustrative only and do not limit the scope of the invention in any way.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Purification of pravastatin

The fermentation broth (100 liters) is acidified to about pH 2.5 to about 5.0 by the addition of sulfuric acid. The acidified fermentation broth was extracted with isobutyl acetate (3 x 50 L). The yield of isobutyl acetate extraction is 95% by HPLC analysis calibrated to an internal standard. HPLC conditions (reversed phase): C ₁₈ column, 5 µm particle size, 150 mm length, 4.6 mm diameter; mobile phase: 45% methanol / water, 0.1% triethylamine, 0.1% glacial acetic acid; flow rate 1.3 ml / min; column temperature 25 ° C; injection volume 10 µl; Ethyl parahydroxybenzoate internal standard; detection: UV λ = 238 nm.

The combined isobutyl acetate phases are extracted with water (35 L) at pH from about 7.5 to about 11.0 by the treated addition of concentrated ammonium hydroxide. The aqueous pravastatin solution obtained is again acidified to a pH of about 2.0 to about 4.0 by addition of 5M sulfuric acid and back extracted with isobutyl acetate (8 liters).

The resulting pravastatin solution in isobutyl acetate was partially dried with pearlite and sodium sulfate. The pravastatin solution was decanted, freed from the drying agent by filtration and decolorized with activated carbon (1.7 g). The solution is filtered to remove charcoal and placed in a vessel equipped with a gas inlet.

Ammonia gas is introduced into the upper space above the solution at 15-25 ° C with rapid stirring. After precipitation appears to be complete, the ammonia feed is stopped and ammonium chloride is added to the mixture to facilitate filtration. The precipitated crystals of pravastatin carboxylate ammonium salt were collected by filtration and washed with isobutyl acetate and then acetone to give the pravastatin ammonium salt of about 94% purity as determined by HPLC with UV detection λ = 238 nm.

Ammonium salt of pravastatin saturated solution: The salt further chloride recrystallized from the following procedure: the active substance is dissolved in isobutyl acetate (96 ml) at about 35-40 ° C. The resulting solution was cooled to containing purification of ammonium pravastatin water (960 mL). Dilute with acetone (96

162 g of solution (ml) and about 30 to 32 ° C and the addition of solid ammonium chloride induces crystallization of pravastatin ammonium salt. Ammonium chloride is added until further addition leads to no apparent crystal growth. After addition of ammonium chloride, the solution is cooled to 0 to 26 ° C. The pravastatin ammonium salt crystals are collected by filtration, washed with isobutyl acetate and acetone as before, and dried at about 40 ° C. Crystals of the ammonium salt of pravastatin (155.5 g) are obtained with about 98% purity as determined by HPLC under the above conditions.

The pravastatin ammonium salt is purified by further recrystallization as follows. The pravastatin ammonium salt (155.5 g active ingredient) was dissolved in water (900 mL). To the resulting solution was added isobutanol (2 mL). The pH of the resulting mixture was raised to about 10 to about 13.7 by addition of concentrated sodium hydroxide solution. The resulting solution was stirred at ambient temperature for 75 minutes and neutralized to pH 7 by the addition of sulfuric acid. The crystallization of pravastatin ammonium salt was induced by the addition of solid ammonium chloride. The crystals (150 g) were collected by filtration and washed with acetone. HPLC analysis of the above conditions indicated that the ammonium salt of pravastatin was obtained with a purity of about 99.3%.

The pravastatin ammonium salt is transsalified into the sodium salt as follows. Pravastatin ammonium salt crystals were dissolved in water (1800 mL). To the aqueous solution was added isobutyl acetate (10.5 L). The resulting solution is acidified to about pH 2 to about 4 by the addition of sulfuric acid, whereby pravastatin is converted back to the free acid. The isobutyl acetate phase containing pravastatin is washed with water (5 x 300 mL). Pravastatin is then converted to the sodium salt and back-extracted to another aqueous phase by vortexing the isobutyl acetate solution with water (900 to 2700 mL), intermittently adding 8M sodium hydroxide until the pH rises to about

7.4 to about 13.

The pravastatin sodium solution is then mixed with an ion exchange resin to capture excess sodium cations. After separation, the aqueous phase was stirred with Amberlite® IRC 50 ion exchange resin in H ⁺ form for 30 minutes at ambient temperature. Stirring is continued until a pH of about 7.4 to about 7.8 is reached.

The solution was then filtered to remove the resin and partially concentrated under reduced pressure to 508 g. The concentrated solution was diluted with acetonitrile (480 mL) to give a solvent mixture of acetonitrile and water 1.4: 1. The solution was stirred with activated carbon (5 g) to decolorize. After filtration of the activated carbon, pravastatin sodium is obtained as crystals (90% yield) by adding additional acetone and acetonitrile to the mixture to give a 1: 3: 12 mixture of water, acetone and acetonitrile (5.9 liters) with cooling to about -10 to about 0 ° C. Pravastatin sodium is obtained in a total yield of 65% with a purity of about 99.3% based on the starting fermented active substance and measured by HPLC under the conditions described above.

Example 2

The procedure is as described in Example 1, but recrystallization from a mixture of water, acetone and acetonitrile is omitted. Lyophilization of a concentrated solution of pravastatin sodium in water gives pravastatin sodium with about 99% purity in about 72% yield. A comparison with the final purity of the product obtained according to Example 1 shows that recrystallization of pravastatin sodium compared to lyophilization results in a slightly purer product.

Examples 3 to 6

The sodium salt of pravastatin is isolated from the fermentation broth as described in Example 1. The yields and purity of the products obtained are shown in Table 1 together with the corresponding organic solvent used for transsalification.

Table 1

Example organic yield purity number solvent (%) (%) 3 dichloromethane 63 96, 6 4 acetate 58 99, 5 5 ethyl formate 51 99, 6 6 butyl methyl ketone 61 99, 5

Example 7

However, following the procedure described in Example 1, in which pravastatin sodium is further purified by repeatedly recrystallizing the ammonium salt of pravastatin, pravastatin sodium is obtained with about 99.6% purity in 58.4% yield.

Example 8

However, following the procedure described in Example 1, in which pravastatin sodium was further purified by repeatedly recrystallizing the ammonium salt of pravastatin, pravastatin sodium was obtained with about 99.8% purity in 53% yield.

Example 9

By the method described in Example 1, the fermentation broth (100 liters) is acidified by the addition of sulfuric acid to a pH of about 2.5 to about 5.0. The acidified fermentation broth was extracted with isobutyl acetate (3 x 50 L). The combined isobutyl acetate phases are then extracted with water (35 liters) at a pH of about 7.5 to about 11.0 by the treated addition of concentrated ammonium hydroxide.

Instead of re-acidification and extraction with isobutyl acetate to give a further enriched organic solution carried out according to the procedure of Example 1, the aqueous extract is concentrated under reduced pressure to a concentration of 140 g / liter. The resulting concentrated solution has a pH of about 4.0 to about 8. The excess ammonia is evaporated.

Ammonium chloride crystals (405 g) were added slowly in portions over 4 hours to the concentrated solution and the pravastatin ammonium salt was allowed to crystallize at ambient temperature.

The crystals were collected by filtration, washed with saturated ammonium chloride solution and added to water (1 liter) at 40 ° C. After dissolution, the temperature was lowered to 30 ° C and ammonium chloride (330 g) was added portionwise over 2 hours. The solution was then stirred at ambient temperature for 15 hours. The crystals of the pravastatin ammonium salt are isolated by filtration, washed with isobutyl acetate and then with acetone and dried. The crystals obtained are further purified by recrystallization, converted into the sodium salt and isolated as described in Example 1. Obtain pravastatin sodium with a purity of about 99.6% in a yield of 64.7%.

Example 10

The procedure of Example 1 is followed, but the pravastatin sodium is recrystallized from a 1: 15 mixture of water and acetone to give the product in total yield, based on the fermented active substance, 64% with 99.6% purity by HPLC.

Example 11

The first two paragraphs of Example 9 are followed. A concentrated aqueous extract (140 g / liter) is obtained. This extract is divided into three equal portions. The concentrated solution is then acidified to about pH 4.0 to about 8.0 by the addition of 1M hydrochloric acid. The procedure of the third paragraph of Example 9 is followed, but the ammonium chloride is replaced by the salt shown in Table 2. The pravastatin salt is precipitated from each of the three portions and converted to the sodium salt.

Table 2

salt purity (%) yield (%) ammonium chloride 99.3 42 sodium chloride 99.4 38 lithium chloride 99.1 34

Claims (42)

PATENT CLAIMS What is claimed is: 1. A method for recovering a sodium salt selected from pravastatin, compactin and lovastatin from a fermentation broth, comprising: (a) a step of forming an enriched solution of the compound; (b) the step of recovering the salt of the compound from the enriched solution; (c) a degree of purification of the salt of the compound; (d) the degree of transsalification of the salt of the compound into the sodium salt of the compound in which it is i) releasing the compound from its salt, ii) contacting the compound with an aqueous sodium hydroxide solution to obtain an aqueous solution of the sodium salt of the compound, and iii) collecting excess sodium ions by contacting the aqueous solution of the sodium salt of the compound with an ion exchange resin. a water-insoluble resin; and (e) a step of isolating the sodium salt of the compound from the aqueous solution of the sodium salt of the compound. Process according to claim 1, characterized in that the enriched solution is an enriched aqueous solution which is prepared by carrying out (a) a step in which the acidic fermentation broth is contacted with an organic extraction solvent to obtain an organic extract of the compound; (b) a step of recovering the organic extract by contacting it with a basic aqueous solution to obtain an aqueous extract; and (c) concentrating the aqueous extract to obtain an enriched aqueous solution of the compound. A process according to claim 1, characterized in that the enriched solution is an enriched organic solution which is prepared by carrying out (a) a step in which the acidic fermentation broth is contacted with an organic extraction solvent to obtain an organic extraction solvent. compound extract; (b) a step of recovering the organic solution by contacting it with a basic aqueous solution to obtain an aqueous extract of the compound; and (c) re-extracting the compound from the aqueous extract by contacting the aqueous extract with an organic re-extraction solvent to obtain an enriched organic solution of the compound. The process according to claim 2, characterized in that the concentration of the aqueous extract is carried out by vacuum distillation. The method of claim 2, wherein the acidic fermentation broth is contacted with an organic extraction solvent at a pH of from about 1 to about 6. 6. The process of claim 2, wherein the back-extraction with a basic aqueous solution is carried out at a pH of from about 7.0 to about 13.7. 7. A method according to claim 2. wherein the organic extraction solvent is selected from the group consisting of C 2 -C 4 alkyl formates, C 1 -C 4 alkyl esters, C 2 -C 4 alkyl carboxylic acids, dichloromethane, dichloroethane, chloroform , carbon tetrachloride and butyl methyl ketone. The process of claim 7, wherein the organic extraction solvent is acetate. The process of claim 3, wherein the acidic fermentation broth is contacted with an organic extraction solvent at a pH of from about 1 to about 6. The process of claim 3, wherein the back-extraction with a basic aqueous solution is carried out at a pH of about 7.0 to about 13.7. The method of claim 3, wherein the re-extraction of the compound with an organic solvent is carried out at a pH of from about 1 to about 6. The process according to claim 3, wherein the organic extraction solvent and the organic re-extraction solvent are each independently selected from the group consisting of C 2 -C 4 alkylformates, C 1 -C 4 alkyl esters, C 1 -C 4 alkyl esters. C 2 -C 4 alkyl, dichloromethane, dichloroethane, chloroform, carbon tetrachloride and butyl methyl ketone. The process according to claim 12, wherein the organic extraction solvent and the organic re-extraction solvent are acetates. 14. The process of claim 13 wherein the organic extraction solvent and the organic re-extraction solvent is isobutyl acetate. 15th The method of claim 1, .... j ú c i s a T ý m. that the degree of recovery salts of the compound performs at at from about -10 to about + 60'C. 16th The method of claim 1, .... j ú c i s a T ý m. that in the recovery stage salts of the compound sol compound it precipitates by adding a metal salt, ammonia, amine or ammonium salt to the enriched solution. The method of claim 16, wherein the salt of the compound is precipitated by the addition of an ammonium salt. The process of claim 17, wherein the ammonium salt is selected from the group consisting of ammonium chloride, NH ₄ Cl; ammonium bromide, NH ₄ Br; ammonium iodide, NH ₄ I; ammonium sulfate, (NH ₄ ) ₂ SO ₄ ; ammonium nitrate, NH ₄ NO ₃ ; ammonium phosphate, (NH ₄ ) ₃ PO ₄ ; ammonium sulfite, (NH ₄ ) ₂ S ₂ O ₄ ; ammonium acetate, NH ₄ OAc and ammonium thiocyanate, NH ₄ SCN. 19. The process of claim 18 wherein the ammonium salt is ammonium chloride. 20. The process of claim 16 wherein the salt of the compound is precipitated by the addition of ammonia gas. The method of claim 16, wherein the salt of the compound is precipitated by the addition of gaseous ammonia and an ammonium salt. The method of claim 16, wherein the salt of the compound is precipitated by the addition of a metal salt. The method of claim 22, wherein the salt is selected from the group consisting of lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, copper salts, iron salts, nickel salts, manganese salts, tin salts, zinc salts and aluminum salts. The method of claim 23, wherein the metal salt is selected from the group consisting of salts containing Li ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Cu ²⁺ , Fe ²⁺ , Fe ions. ³⁺ , Ni ²⁺ , Mn ²⁺ , Sn ²⁺ , Zn ^2+, and Al ³⁺ . The method of claim 16, wherein the salt of the compound is precipitated by the addition of an amine salt. 26. The process of claim 25 wherein the amine salt comprises an amine moiety selected from the group consisting of primary, secondary, and tertiary alkylamines having from 1 to 3 carbon atoms in each of the alkyl moieties. The method of claim 1, wherein the step of purifying the salt of the compound comprises the step of recrystallizing the salt of the compound. 28. The method of claim 27, wherein recrystallizing the salt of the compound comprises (A) a step of dissolving the salt of the compound in water to obtain an aqueous solution of the salt of the compound; (B) a step of diluting the aqueous salt solution with an anti-solvent; and compound (C) a step of adding a salt containing the same metal, ammonium ion or amine ion as the compounds. ion salt 29th 10. The method of claim 28, wherein the method is: c i s a wherein the anti-solvent is selected from the group consisting of ethyl acetate, isobutyl acetate, isobutanol, acetone, acetonitrile, and ethanol. The method of claim 28, wherein the salt containing the same metal ion, ammonium ion or amine ion as the salt of the compound is an ammonium salt. The method of claim 30, wherein the ammonium salt is selected from the group consisting of ammonium chloride, NH ₄ Cl; ammonium bromide, NH ₄ Br; ammonium iodide, NH ₄ I; ammonium sulfate, (NH ₄ ) ₂ SO ₄ ; ammonium nitrate, NH ₄ NO ₃ ; ammonium phosphate, (NH ₄ ) ₃ PO ₄ ; ammonium sulfite, (NH ₄ ) ₂ S ₂ O ₄ ; ammonium acetate, NH ₄ OAc and ammonium thiocyanate, NH ₄ SCN. 32. The process of claim 31 wherein the ammonium salt is ammonium chloride. The method of claim 1, wherein the release of the compound from the salt of the compound comprises (a) a step of dissolving the salt of the compound in an aqueous solvent, (b) a step of adding the protic acid to the aqueous solvent, and an organic solvent to form an organic solution of the compound, further contacting the compound with an aqueous sodium hydroxide solution by back-extracting the organic solution of the compound with an aqueous sodium hydroxide solution. 34. The process of claim 33, wherein the extraction of the compound is carried out at a pH of from about 1 to about 6. 35. The process of claim 33, wherein the organic solvent is selected from the group consisting of alkyl formates having 2 to 4 carbon atoms in the alkyl moiety, alkyl alkanoates having 1 to 4 carbon atoms in the alkyl moiety and 2 to 4 carbon atoms in the alkane moiety. , dichloromethane, dichloroethane, chloroform, carbon tetrachloride and butylmethylketone. 36. The process of claim 35, wherein the organic solvent is isobutyl acetate. The method of claim 33, wherein the back extraction of the organic solution of the compound into an aqueous sodium hydroxide solution is carried out at a pH of from about 6.5 to about 13.7. The method of claim 1, wherein the step of isolating the sodium salt of the compound comprises (a) a step of concentrating the aqueous solution of the sodium salt of the compound and (b) diluting the aqueous solution of the sodium salt of the compound with an organic solvent or organic solvent mixture. 39. The method of claim 1, wherein the step of isolating the sodium salt of the compound comprises the step of lyophilizing the aqueous solution of the sodium salt of the compound. 40. The process of claim 38, wherein the step of concentrating the aqueous solution of the sodium salt of the compound is performed by vacuum distillation. 41. The method of claim 38, wherein the step of concentrating the aqueous solution of the sodium salt of the compound is performed by nanofiltration. The method of claim 38, v y z načuj ú c i sa by aqueous sodium solution salt compound the diluted acetone. The method of claim 38, v y z načuj ú c i sa by aqueous sodium solution salt compound the diluted an organic solvent mixture selected from File consisting of a mixture of acetone and acetonitrile, a mixture of ethanol and acetonitrile, and a mixture of ethanol and ethyl acetate. 44. The method of claim 38, wherein the step of isolating the sodium salt of the compound is carried out at a temperature of from about -10 ° C to about 160 ° C. 45. Pravastatin sodium which is substantially free of pravastatin lactone. 46. The pravastatin sodium of claim 45, which comprises 0.5 wt. or less total impurity. 47. The pravastatin sodium of claim 46, which comprises 0.2 wt. or less total impurity.